The present invention relates to a process for increasing the FVII (Factor VII) sensitivity of a thromboplastin reagent by means of heat treatment.
Quick""s thromboplastin time (PT) is employed as a test for screening for a deficiency in coagulation factors in patient blood. The PT is also used to monitor therapy with oral anticoagulants. The preferred PT for normal blood donors is 10-14 seconds. The preferred PT for factor VII-deficient plasmas should be more than 60 seconds. The FVII sensitivity is therefore defined as the ratio of the coagulation time of the FVII-deficient plasma and the coagulation time for normal blood donors.
The invention describes a process for increasing the factor VII sensitivity of thromboplastin reagents by increasing the PT for factor VII-deficient plasmas without significantly altering the PT for normal plasmas.
Active thromboplastin induces coagulation in plasma and is composed of a lipid component and a protein component. The protein, i.e. tissue factor, is membrane-bound and is found in many different tissues. The binding between the protein and the lipid is based on hydrophobic interactions and is Ca2+-independent. The protein moiety is composed of a glycoprotein having a molecular weight of 43-53 kDa. One molecule of tissue factor is able to bind to one molecule of FVII or FVIIa. The binding of FVII/FVIIa to tissue factor is Ca2+-dependent. The complex composed of lipid, tissue factor and FVIIa cleaves FX to form FXa and thereby finally elicits coagulation by means of activating prothrombin.
The onset of coagulation in a plasma at from 10 to 14 sec. after adding a thromboplastin reagent indicates that the coagulation system is intact. An increase in the coagulation time points to an impairment. Impairments can occur as a result of the concentrations of one or more coagulation factors being too low. Thromboplastin reagents of varying origin frequently differ in their sensitivity for indicating the deficiency of particular factors. This can sometimes be due to the carry-over of small quantities of the factors to be determined into the reagent. Common methods for specifically removing small quantities of protein are, inter alia, immunoadsorption or, in the case of the vitamin K-dependent coagulation factors such as FII, VII, IX and X, barium sulfate adsorption (WO 90/05740). In the case of reagents of human origin, it is frequently only the sensitivity with regard to FVII which is inadequate.
Proteases, i.e. factors VIIa, IXa, Xa, XIa and XIIa, and plasma kallikrein and thrombin, are essential factors of the coagulation cascade.
Disadvantages of the previously described methods are, firstly, that FVII binds to tissue factor and the yield of tissue factor which is achieved by such an adsorption method is therefore reduced and, secondly, that both methods demand a substantial input of equipment and time, particularly on a production scale.
In the present invention, it is demonstrated that it is surprisingly possible, under defined conditions, to selectively inhibit the residual FVIIa activity of the reagent and consequently to arrive at the desired result in a simple manner without impairing the tissue factor or other factors of the coagulation cascade. The ratio, which is termed FVII sensitivity, of the coagulation time of the FVII-deficient plasma to the coagulation time of a normal plasma is preferably  greater than 4, particularly preferably  greater than 6 and very particularly preferably  greater than 10.
Factor VIIa possesses a protease activity and belongs to the serine protease class, as do the other coagulation factors IXa, Xa, XIa, XIIa, plasma kallikrein and thrombin. Another feature common to these coagulation factors is that they are initially present in zymogenic form and protein bonds have to be cleaved for their protease activity to be displayed.
A large number of protease inhibitors which at least inhibit the active forms of these zymogens, e.g. thrombin, have been described (W.B. Lawson et al., 1982, Folia Haematol. Leipzig 109 (1982) 1, pp. 52-60). They include sulfonyl fluorides, such as phenylmethylsulfonyl fluoride (PMSF), p-aminoethylbenzenesulfonyl fluoride (AEBSF) and 4-aminophenylmethanesulfonyl fluoride (p-APMSF), organofluorophosphates, such as diisopropyl fluorophosphate (DFP), chloromethyl ketones and also peptides, such as leupeptins or proteins of the serpin family, such as C1 inhibitor, antithrombin III and aprotinin.
The protease inhibitors suffer from the disadvantage that, in the PT, they inhibit the coagulation factors IXa, Xa, XIa, XIIa, plasma kallikrein and thrombin in the sample to be determined in addition to inhibiting the factor FVII or FVIIa which has been carried over. On this basis, they would prolong coagulation time and give a false impression of a pathological value. It is to be expected, therefore, that these inhibitors would not be suitable for being used in a PT reagent. Surprisingly, however, it is possible to employ these inhibitors under specific conditions without prolonging the coagulation times of the sample; however, the FVII sensitivity of the reagent is nevertheless improved.
Another option for inhibiting the enzymic activity of a protein is that of preparing specific antibodies which inhibit the active center of the protein. It is possible to obtain a variety of antibodies against coagulation factor VII, including at least one polyclonal antibody which inhibits the enzymic activity of FVII/FVIIa. Simply adding the antibody to a PT reagent would be disadvantageous since the antibody also inhibits the FVII/FVIIa of the sample. In the present invention, it is demonstrated that there is, surprisingly, a concentration range for adding the anti-FVII antibody in which the coagulation time of a normal plasma is not prolonged but that of an FVII-deficient plasma is prolonged markedly, such that the FVII sensitivity of the reagent is substantially increased.
It has also been previously reported that serine pro-teases can be oxidatively inhibited (S. E. Lind et al., 1993, Blood 82, 5 15522-1531). This method has the disadvantage that the fatty acids of the phospholipids can be oxidized in addition to the disadvantage that the coagulation cascade serine proteases in the sample are inhibited (Dasgupta, A. et al., Live Science 1992, 50, 875-882). The oxidation products which are produced in this connection likewise inhibit the PT (T. W. Barrowcliffe et al., 1975, Thrombos. Diathes. haemorrh. 33, 271).
Surprisingly, however, the oxidation process can be controlled such that it is possible to disregard the undesirable side reactions, i.e. while the coagulation time of normal plasmas is not prolonged, the FVII sensitivity of the reagent is improved. Suitable oxidizing agents are hypochlorite, hydrogen peroxide, permanganate and manganese dioxide. Many antioxidants, such as ascorbic acid, thiols, such as glutathione, acetyl cysteine, dithioerythreitol and tocopherols and tocopherol derivatives, such as di-tert-butyl-p-hydroxyanisole (BHA), di-tert-butyl-p-cresol (BHT), Trolox and propylgallate, are able to exert an oxidative effect in the presence of metal ions. The metal ions which catalyze the oxidation include, in particular, iron, copper and zinc. The metal-catalyzed oxidation is particularly suitable for inhibiting residual FVII/FVIIa activities.
The process of the present invention uses simple methods for heat-treating the tissue extract selectively; without thereby restricting ourselves to one particular reaction mechanism, it might be possible to explain the efficacy of the process on the basis that the specific heat treatment removes residual FVII/FVIIa activities. The heat treatment is advantageously carried out for a short time at high temperatures.
At low temperatures of between +40 and +60xc2x0 C., inactivation of the FVII activities depends on a number of factors, inter alia the dimensions and the thermal conductivity of the vessel employed and the solution used, some of which cannot be controlled. The heating time can also vary widely. The heating temperature is advantageously between +65xc2x0 C. and +160xc2x0 C., preferably between +80xc2x0 C. and 140xc2x0 C., particularly preferably between +90 and +120xc2x0 C.
So that the liquid which is supplied is heated to the relevant heating temperature in as short a time as possible and can then be cooled down again in a similarly short period of time, the apparatus in which the heating is carried out is expediently equipped with at least one connection for heating medium and at least one connection for cooling medium. The apparatus advantageously comprises three regions or sections: a heating section, in which the reagent is heated to the requisite temperature, a holding section, in which the reagent is held at this temperature for a specified time, and a cooling section in which the reagent is cooled down once again to a low temperature.
The construction of the heat-transfer apparatus should be such that the difference between the temperature to which the solution is heated and the temperature of the heating medium is as small as possible, as a consequence of which the product is spared to the greatest possible extent on account of the resulting low excess temperature of the walls which are in contact with the product.
Preference is given to a process for continuously heating for a short time, as described in EP-A 0 571 771 for virus inactivation.
The heating and/or cooling time should in each case be less than 30 seconds, preferably, however, less than 5 seconds. The holding time can be between 0.1 and 30 seconds, preferably, however, between 0.5 and 20 seconds.
Without restricting the invention to the example of human placenta extract, the following example demonstrates the inactivation of residual FVII/FVIIa activities by heating, as employed for a Behringwerke AG tissue thromboplastin-containing Quick reagent THROMBOREL S, Prod. No. OUHP placenta-thromboplastin. Preservatives, antioxidants, carbohydrates, proteins and amino acids, and combinations thereof, are advantageously added to the reagent as stabilizers. In the individual substance classes, the following compounds are particularly suitable:
Preservatives:
Mergal K9N (5-chlor-2-methylisothiazolin 3-on-2-methylisothiazolin 3-o), sodium azide phenol, amphotericin, gentamicin, piperacilin and ciprofloxacin.
Antioxidants:
Di-tert-butyl-p-hydroxyanisole (BHA), di-tert-butyl-p-cresol (BHT), spermine, propygallate , tocopherols,
Trolox (6-hydroxy-2,5,7,8 tetramethyl-chroman-2-carboxylic acid, dithioerythreitol and glutathione.
Carbohydrates:
Trehalose, sucrose, Tylose (methylcellulose), mannitol, xanthan gum, phytagel, carrageenan and polyethylene glycol.
Proteins:
Lactoglobulin, lipoproteins and albumins, such as serum albumin, lactalbumin and ovalbumin.
Amino acids:
Acetylcysteine, acetylmethionine, glycine, lysine, histidine and serine.
The present invention also relates to processes for Increasing the FVII sensitivity of thromboplastin reagents, which comprise selectively inhibiting the residual FVII/FVlla activity in the reagent, wherein a solution of these reagents is heated to greater than 65xc2x0 C., and wherein the thromboplastin reagent is recombinant tissue thromboplastin or a biologically active variant of a tissue thromboplastin.